Phylogenetic and Chemical Diversity of a Hybrid-Isoprenoid-Producing Streptomycete Lineage

ABSTRACTStreptomycesspecies dedicate a large portion of their genomes to secondary metabolite biosynthesis. A diverse and largely marine-derived lineage within this genus has been designated MAR4 and identified as a prolific source of hybrid isoprenoid (HI) secondary metabolites. These terpenoid-containing compounds are common in nature but rarely observed as bacterial secondary metabolites. To assess the phylogenetic diversity of the MAR4 lineage, complementary culture-based and culture-independent techniques were applied to marine sediment samples collected off the Channel Islands, CA. The results, including those from an analysis of publically available sequence data and strains isolated as part of prior studies, placed 40 new strains in the MAR4 clade, of which 32 originated from marine sources. When combined with sequences cloned from environmental DNA, 28 MAR4 operational taxonomic units (0.01% genetic distance) were identified. Of these, 82% consisted exclusively of either cloned sequences or cultured strains, supporting the complementarity of these two approaches. Chemical analyses of diverse MAR4 strains revealed the production of five different HI structure classes. All 21 MAR4 strains tested produced at least one HI class, with most strains producing from two to four classes. The two major clades within the MAR4 lineage displayed distinct patterns in the structural classes and the number and amount of HIs produced, suggesting a relationship between taxonomy and secondary metabolite production. The production of HI secondary metabolites appears to be a phenotypic trait of the MAR4 lineage, which represents an emerging model with which to study the ecology and evolution of HI biosynthesis.

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Phylogenetic Distribution of Secondary Metabolites in the Bacillus subtilis Species Complex

mSystems ◽

10.1128/msystems.00057-21 ◽

2021 ◽

Vol 6 (2) ◽

Author(s):

Kat Steinke ◽

Omkar S. Mohite ◽

Tilmann Weber ◽

Ákos T. Kovács

Keyword(s):

Bacillus Subtilis ◽

Secondary Metabolites ◽

Secondary Metabolite ◽

Species Complex ◽

Gene Clusters ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Content Type ◽

Frameshift Mutations ◽

Metabolite Production

ABSTRACT Microbes produce a plethora of secondary (or specialized) metabolites that, although not essential for primary metabolism, benefit them to survive in the environment, communicate, and influence cell differentiation. Biosynthetic gene clusters (BGCs), responsible for the production of these secondary metabolites, are readily identifiable on bacterial genome sequences. Understanding the phylogeny and distribution of BGCs helps us to predict the natural product synthesis ability of new isolates. Here, we examined 310 genomes from the Bacillus subtilis group, determined the inter- and intraspecies patterns of absence/presence for all BGCs, and assigned them to defined gene cluster families (GCFs). This allowed us to establish patterns in the distribution of both known and unknown products. Further, we analyzed variations in the BGC structures of particular families encoding natural products, such as plipastatin, fengycin, iturin, mycosubtilin, and bacillomycin. Our detailed analysis revealed multiple GCFs that are species or clade specific and a few others that are scattered within or between species, which will guide exploration of the chemodiversity within the B. subtilis group. Surprisingly, we discovered that partial deletion of BGCs and frameshift mutations in selected biosynthetic genes are conserved within phylogenetically related isolates, although isolated from around the globe. Our results highlight the importance of detailed genomic analysis of BGCs and the remarkable phylogenetically conserved erosion of secondary metabolite biosynthetic potential in the B. subtilis group. IMPORTANCE Members of the B. subtilis species complex are commonly recognized producers of secondary metabolites, among those, the production of antifungals, which makes them promising biocontrol strains. While there are studies examining the distribution of well-known secondary metabolites in Bacilli, intraspecies clade-specific distribution has not been systematically reported for the B. subtilis group. Here, we report the complete biosynthetic potential within the B. subtilis group to explore the distribution of the biosynthetic gene clusters and to reveal an exhaustive phylogenetic conservation of secondary metabolite production within Bacillus that supports the chemodiversity within this species complex. We identify that certain gene clusters acquired deletions of genes and particular frameshift mutations, rendering them inactive for secondary metabolite biosynthesis, a conserved genetic trait within phylogenetically conserved clades of certain species. The overview guides the assignment of the secondary metabolite production potential of newly isolated Bacillus strains based on genome sequence and phylogenetic relatedness.

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Rare unclassified 16S rRNA operational taxonomic units from the uncharted Engaño Bay (Argentinean Patagonia)

Canadian Journal of Microbiology ◽

10.1139/cjm-2017-0342 ◽

2018 ◽

Vol 64 (1) ◽

pp. 91-96

Author(s):

Andrea Y. Calvo ◽

Julieta M. Manrique ◽

Leandro R. Jones

Keyword(s):

16S Rrna ◽

Community Dynamics ◽

Sequence Data ◽

River Estuary ◽

Fragmented Habitats ◽

Operational Taxonomic Units ◽

Microbial Community Dynamics ◽

Argentinean Patagonia ◽

Culture Independent ◽

Taxonomic Assignments

Rare microbes make up most of the diversity of marine microbiomes, and recent works have highlighted their importance for microbial community dynamics and in fragmented habitats. Rare taxa have been infrequently studied in comparison with abundant groups, and rare unclassified sequences are common in culture-independent studies. Here, we describe a detailed analysis of nonclassifiable sequences from the Chubut river estuary at the Argentinean Patagonia. Standard taxonomic assignments of environmental 16S rRNA sequences resulted in about 13% unclassified operational taxonomic units (OTUs). The potential affiliations of these OTUs could be narrowed by mapping the classification software assignments on a phylogeny obtained directly from our environmental sequence data. Customized BLAST analyses were remarkably consistent with these phylogenetic assignments, especially when the unclassified OTUs were blasted against sequences from cultured and type microorganisms. In addition, our BLAST analyses revealed significant similarities between several unclassified OTUs and a plethora of unclassified sequences from around the world. Further phylogenetic comparisons with 6194 carefully selected reference sequences showed that these unclassified sequences may correspond to 5 unnamed groups, possibly encompassing ranks from subclass to family inside the Alphaproteobacteria, and to an unknown Gracilibacteria lineage. Overall, these results demonstrate the value of straight phylogenetic analysis, customized BLAST searches, and comparisons with sequences from type material, for the systematic study of rare unclassified sequences.

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An IgaA/UmoB Family Protein fromSerratia marcescensRegulates Motility, Capsular Polysaccharide Biosynthesis, and Secondary Metabolite Production

Applied and Environmental Microbiology ◽

10.1128/aem.02575-17 ◽

2018 ◽

Vol 84 (6) ◽

Cited By ~ 10

Author(s):

Nicholas A. Stella ◽

Kimberly M. Brothers ◽

Jake D. Callaghan ◽

Angelina M. Passerini ◽

Cihad Sigindere ◽

...

Keyword(s):

Secondary Metabolites ◽

Secondary Metabolite ◽

Intracellular Signaling ◽

Biofilm Formation ◽

Capsular Polysaccharide ◽

Biologically Active ◽

Public Health Importance ◽

Content Type ◽

Polysaccharide Biosynthesis ◽

Transcriptional Changes

ABSTRACTSecondary metabolites are an important source of pharmaceuticals and key modulators of microbe-microbe interactions. The bacteriumSerratia marcescensis part of theEnterobacteriaceaefamily of eubacteria and produces a number of biologically active secondary metabolites. In this study, we screened for novel regulators of secondary metabolites synthesized by a clinical isolate ofS. marcescensand found mutations in a gene for an uncharacterized UmoB/IgaA family member here namedgumB. Mutation ofgumBconferred a severe loss of the secondary metabolites prodigiosin and serratamolide. ThegumBmutation conferred pleiotropic phenotypes, including altered biofilm formation, highly increased capsular polysaccharide production, and loss of swimming and swarming motility. These phenotypes corresponded to transcriptional changes infimA,wecA, andflhD. Unlike other UmoB/IgaA family members,gumBwas found to be not essential for growth inS. marcescens, yetigaAfromSalmonella enterica,yrfFfromEscherichia coli, and an uncharacterized predicted ortholog fromKlebsiella pneumoniaecomplemented thegumBmutant secondary metabolite defects, suggesting highly conserved function. These data support the idea that UmoB/IgaA family proteins are functionally conserved and extend the known regulatory influence of UmoB/IgaA family proteins to the control of competition-associated secondary metabolites and biofilm formation.IMPORTANCEIgaA/UmoB family proteins are found in members of theEnterobacteriaceaefamily of bacteria, which are of environmental and public health importance. IgaA/UmoB family proteins are thought to be inner membrane proteins that report extracellular stresses to intracellular signaling pathways that respond to environmental challenge. This study introduces a new member of the IgaA/UmoB family and demonstrates a high degree of functional similarity between IgaA/UmoB family proteins. Moreover, this study extends the phenomena controlled by IgaA/UmoB family proteins to include the biosynthesis of antimicrobial secondary metabolites.

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Response of Secondary Metabolism of Hypogean Actinobacterial Genera to Chemical and Biological Stimuli

Applied and Environmental Microbiology ◽

10.1128/aem.01125-18 ◽

2018 ◽

Vol 84 (19) ◽

Cited By ~ 5

Author(s):

Brett C. Covington ◽

Jeffrey M. Spraggins ◽

Audrey E. Ynigez-Gutierrez ◽

Zachary B. Hylton ◽

Brian O. Bachmann

Keyword(s):

Secondary Metabolites ◽

Small Molecules ◽

Mixed Culture ◽

Secondary Metabolite ◽

Biologically Active ◽

Secondary Metabolite Production ◽

Microbial Culture ◽

Content Type ◽

Metabolite Production ◽

Comparative Metabolomics

ABSTRACT Microorganisms within microbial communities respond to environmental challenges by producing biologically active secondary metabolites, yet the majority of these small molecules remain unidentified. We have previously demonstrated that secondary metabolite biosynthesis in actinomycetes can be activated by model environmental chemical and biological stimuli, and metabolites can be identified by comparative metabolomics analyses under different stimulus conditions. Here, we surveyed the secondary metabolite productivity of a group of 20 phylogenetically diverse actinobacteria isolated from hypogean (cave) environments by applying a battery of stimuli consisting of exposure to antibiotics, metals, and mixed microbial culture. Comparative metabolomics was used to reveal secondary metabolite responses from stimuli. These analyses revealed substantial changes in global metabolomic dynamics, with over 30% of metabolomic features increasing more than 10-fold under at least one stimulus condition. Selected features were isolated and identified via nuclear magnetic resonance (NMR), revealing several known secondary metabolite families, including the tetarimycins, aloesaponarins, hypogeamicins, actinomycins, and propeptins. One prioritized metabolite was identified to be a previously unreported aminopolyol polyketide, funisamine, produced by a cave isolate of Streptosporangium when exposed to mixed culture. The production of funisamine was most significantly increased in mixed culture with Bacillus species. The biosynthetic gene cluster responsible for the production of funisamine was identified via genomic sequencing of the producing strain, Streptosporangium sp. strain KDCAGE35, which facilitated a deduction of its biosynthesis. Together, these data demonstrate that comparative metabolomics can reveal the stimulus-induced production of natural products from diverse microbial phylogenies. IMPORTANCE Microbial secondary metabolites are an important source of biologically active and therapeutically relevant small molecules. However, much of this active molecular diversity is challenging to access due to low production levels or difficulty in discerning secondary metabolites within complex microbial extracts prior to isolation. Here, we demonstrate that ecological stimuli increase secondary metabolite production in phylogenetically diverse actinobacteria isolated from understudied hypogean environments. Additionally, we show that comparative metabolomics linking stimuli to metabolite response data can effectively reveal secondary metabolites within complex biological extracts. This approach highlighted secondary metabolites in almost all observed natural product classes, including low-abundance analogs of biologically relevant metabolites, as well as a new linear aminopolyol polyketide, funisamine. This study demonstrates the generality of activating stimuli to potentiate secondary metabolite production across diverse actinobacterial genera.

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Lysobacter enzymogenes Uses Two Distinct Cell-Cell Signaling Systems for Differential Regulation of Secondary-Metabolite Biosynthesis and Colony Morphology

Applied and Environmental Microbiology ◽

10.1128/aem.01841-13 ◽

2013 ◽

Vol 79 (21) ◽

pp. 6604-6616 ◽

Cited By ~ 49

Author(s):

Guoliang Qian ◽

Yulan Wang ◽

Yiru Liu ◽

Feifei Xu ◽

Ya-Wen He ◽

...

Keyword(s):

Secondary Metabolites ◽

Cell Signaling ◽

Secondary Metabolite ◽

Colony Morphology ◽

Content Type ◽

Signaling Systems ◽

Lysobacter Enzymogenes ◽

Bioactive Secondary Metabolites ◽

Secondary Metabolite Biosynthesis ◽

First Time

ABSTRACTLysobacter enzymogenesis a ubiquitous environmental bacterium that is emerging as a potentially novel biological control agent and a new source of bioactive secondary metabolites, such as the heat-stable antifungal factor (HSAF) and photoprotective polyene pigments. Thus far, the regulatory mechanism(s) for biosynthesis of these bioactive secondary metabolites remains largely unknown inL. enzymogenes. In the present study, the diffusible signal factor (DSF) and diffusible factor (DF)-mediated cell-cell signaling systems were identified for the first time fromL. enzymogenes. The results show that both Rpf/DSF and DF signaling systems played critical roles in modulating HSAF biosynthesis inL. enzymogenes. Rpf/DSF signaling and DF signaling played negative and positive effects in polyene pigment production, respectively, with DF playing a more important role in regulating this phenotype. Interestingly, only Rpf/DSF, but not the DF signaling system, regulated colony morphology ofL. enzymgenes. Both Rpf/DSF and DF signaling systems were involved in the modulation of expression of genes with diverse functions inL. enzymogenes, and their own regulons exhibited only a few loci that were regulated by both systems. These findings unveil for the first time new roles of the Rpf/DSF and DF signaling systems in secondary metabolite biosynthesis ofL. enzymogenes.

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Draft Genome Sequence and Secondary Metabolite Biosynthetic Potential of the Lysobacter niastensis Type Strain DSM 18481

Microbiology Resource Announcements ◽

10.1128/mra.01296-20 ◽

2021 ◽

Vol 10 (1) ◽

Author(s):

Paolo Turrini ◽

Irene Artuso ◽

Marco Tescari ◽

Gabriele Andrea Lugli ◽

Emanuela Frangipani ◽

...

Keyword(s):

Secondary Metabolites ◽

Small Molecules ◽

Secondary Metabolite ◽

Genome Sequence ◽

Type Strain ◽

Draft Genome ◽

Gene Clusters ◽

Draft Genome Sequence ◽

Content Type

ABSTRACT Lysobacter niastensis belongs to a group of bacterial predators that produce a number of bioactive small molecules endowed with lytic properties toward other microorganisms. Here, we report the draft genome sequence of the type strain DSM 18481 and the identification of gene clusters implicated in the biosynthesis of secondary metabolites.

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Gliotoxin, a Known Virulence Factor in the Major Human Pathogen Aspergillus fumigatus, Is Also Biosynthesized by Its Nonpathogenic Relative Aspergillus fischeri

mBio ◽

10.1128/mbio.03361-19 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 3

Author(s):

Sonja L. Knowles ◽

Matthew E. Mead ◽

Lilian Pereira Silva ◽

Huzefa A. Raja ◽

Jacob L. Steenwyk ◽

...

Keyword(s):

Secondary Metabolites ◽

Aspergillus Fumigatus ◽

Secondary Metabolite ◽

Virulence Factors ◽

Secondary Metabolite Production ◽

Human Pathogen ◽

Content Type ◽

Metabolite Production ◽

Aspergillus Fischeri ◽

Similarities And Differences

ABSTRACT Aspergillus fumigatus is a major opportunistic human pathogen. Multiple traits contribute to A. fumigatus pathogenicity, including its ability to produce specific secondary metabolites, such as gliotoxin. Gliotoxin is known to inhibit the host immune response, and genetic mutants that inactivate gliotoxin biosynthesis (or secondary metabolism in general) attenuate A. fumigatus virulence. The genome of Aspergillus fischeri, a very close nonpathogenic relative of A. fumigatus, contains a biosynthetic gene cluster that is homologous to the A. fumigatus gliotoxin cluster. However, A. fischeri is not known to produce gliotoxin. To gain further insight into the similarities and differences between the major pathogen A. fumigatus and the nonpathogen A. fischeri, we examined whether A. fischeri strain NRRL 181 biosynthesizes gliotoxin and whether the production of secondary metabolites influences the virulence profile of A. fischeri. We found that A. fischeri biosynthesizes gliotoxin under the same conditions as A. fumigatus. However, whereas loss of laeA, a master regulator of secondary metabolite production (including gliotoxin biosynthesis), has previously been shown to reduce A. fumigatus virulence, we found that laeA loss (and loss of secondary metabolite production) in A. fischeri does not influence its virulence. These results suggest that LaeA-regulated secondary metabolites are virulence factors in the genomic and phenotypic background of the major pathogen A. fumigatus but are much less important in the background of the nonpathogen A. fischeri. Understanding the observed spectrum of pathogenicity across closely related pathogenic and nonpathogenic Aspergillus species will require detailed characterization of their biological, chemical, and genomic similarities and differences. IMPORTANCE Aspergillus fumigatus is a major opportunistic fungal pathogen of humans, but most of its close relatives are nonpathogenic. Why is that so? This important, yet largely unanswered, question can be addressed by examining how A. fumigatus and its close nonpathogenic relatives are similar or different with respect to virulence-associated traits. We investigated whether Aspergillus fischeri, a nonpathogenic close relative of A. fumigatus, can produce gliotoxin, a mycotoxin known to contribute to A. fumigatus virulence. We discovered that the nonpathogenic A. fischeri produces gliotoxin under the same conditions as those of the major pathogen A. fumigatus. However, we also discovered that, in contrast to what has previously been observed in A. fumigatus, the loss of secondary metabolite production in A. fischeri does not alter its virulence. Our results are consistent with the “cards of virulence” model of opportunistic fungal disease, in which the ability to cause disease stems from the combination (“hand”) of virulence factors (“cards”) but not from individual factors per se.

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Analysis of Endophyte Diversity of Rheum palmatum from Different Production Areas in Gansu Province of China and the Association with Secondary Metabolite

Microorganisms ◽

10.3390/microorganisms9050978 ◽

2021 ◽

Vol 9 (5) ◽

pp. 978

Author(s):

Dawei Chen ◽

Lingyun Jia ◽

Qinzheng Hou ◽

Xiang Zhao ◽

Kun Sun

Keyword(s):

Secondary Metabolites ◽

Secondary Metabolite ◽

Endophytic Fungi ◽

High Throughput Sequencing ◽

Gansu Province ◽

Operational Taxonomic Units ◽

Rheum Palmatum ◽

Microbe Interactions ◽

Production Area ◽

Production Areas

Investigations of the differences in the metabolites of medicinal plants have typically focused on the effects of external environmental factors. However, little is known about the relationship between endophytes diversity and host metabolites. We used high-throughput sequencing methods to compare the endophyte diversity of Rheum palmatum from eight different production areas in Gansu Province of China and to analyze the association between those areas and five secondary metabolites (aloe-emodin, rhein, emodin, chrysophanol, and physcion). The results show that the diversity and OTUs (Operational taxonomic units) abundance of endophytic fungi and bacteria of R. palmatum differed according to production area. Spearman analysis showed that the five secondary metabolites of R. palmatum were positively correlated with the diversity and abundance of endophytic fungi. Comparing both space and environmental differences to determine influences on community structure, VPA analysis revealed that geographic factors explained more difference in community composition of fungal and bacterial endophytes than climate factors. PICRUSt and FUNGuild predictive analysis indicated that metabolites were the primary components of endophytic bacteria in all samples, while the function of endophytic fungi was composed of dominant trophic modes (saprotroph and pathotroph), and relative abundances were different. Our results help elucidate the correlation of plant–microbe interactions and offer pivotal information to reveal the role of endophytes in the production of R. palmatum and its important secondary metabolite.

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Genomic and Chemical Diversity of Bacillus subtilis Secondary Metabolites against Plant Pathogenic Fungi

mSystems ◽

10.1128/msystems.00770-20 ◽

2021 ◽

Vol 6 (1) ◽

Cited By ~ 1

Author(s):

Heiko T. Kiesewalter ◽

Carlos N. Lozano-Andrade ◽

Mario Wibowo ◽

Mikael L. Strube ◽

Gergely Maróti ◽

...

Keyword(s):

Bacillus Subtilis ◽

Secondary Metabolites ◽

Pathogenic Fungus ◽

Gene Clusters ◽

Chemical Diversity ◽

Biosynthetic Gene ◽

Nonribosomal Peptide ◽

Content Type ◽

Wide Range

ABSTRACT Bacillus subtilis produces a wide range of secondary metabolites providing diverse plant growth-promoting and biocontrol abilities. These secondary metabolites include nonribosomal peptides with strong antimicrobial properties, causing either cell lysis, pore formation in fungal membranes, inhibition of certain enzymes, or bacterial protein synthesis. However, the natural products of B. subtilis are mostly studied either in laboratory strains or in individual isolates, and therefore, a comparative overview of secondary metabolites from various environmental B. subtilis strains is missing. In this study, we isolated 23 B. subtilis strains from 11 sampling sites, compared the fungal inhibition profiles of wild types and their nonribosomal peptide mutants, followed the production of targeted lipopeptides, and determined the complete genomes of 13 soil isolates. We discovered that nonribosomal peptide production varied among B. subtilis strains coisolated from the same soil samples. In vitro antagonism assays revealed that biocontrol properties depend on the targeted plant pathogenic fungus and the tested B. subtilis isolate. While plipastatin alone is sufficient to inhibit Fusarium spp., a combination of plipastatin and surfactin is required to hinder growth of Botrytis cinerea. Detailed genomic analysis revealed that altered nonribosomal peptide production profiles in specific isolates are due to missing core genes, nonsense mutation, or potentially altered gene regulation. Our study combines microbiological antagonism assays with chemical nonribosomal peptide detection and biosynthetic gene cluster predictions in diverse B. subtilis soil isolates to provide a broader overview of the secondary metabolite chemodiversity of B. subtilis. IMPORTANCE Secondary or specialized metabolites with antimicrobial activities define the biocontrol properties of microorganisms. Members of the Bacillus genus produce a plethora of secondary metabolites, of which nonribosomally produced lipopeptides in particular display strong antifungal activity. To facilitate the prediction of the biocontrol potential of new Bacillus subtilis isolates, we have explored the in vitro antifungal inhibitory profiles of recent B. subtilis isolates, combined with analytical natural product chemistry, mutational analysis, and detailed genome analysis of biosynthetic gene clusters. Such a comparative analysis helped to explain why selected B. subtilis isolates lack the production of certain secondary metabolites.

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Beyond Toxin Transport: Novel Role of ABC Transporter for Enzymatic Machinery of Cereulide NRPS Assembly Line

mBio ◽

10.1128/mbio.01577-20 ◽

2020 ◽

Vol 11 (5) ◽

Author(s):

A. Gacek-Matthews ◽

Z. Chromiková ◽

M. Sulyok ◽

G. Lücking ◽

I. Barák ◽

...

Keyword(s):

Natural Products ◽

Secondary Metabolites ◽

Cell Membrane ◽

Secondary Metabolite ◽

Abc Transporter ◽

Molecular Mechanisms ◽

Host Cells ◽

Nonribosomal Peptide ◽

Content Type ◽

Direct Involvement

ABSTRACT Nonribosomal peptide synthetases (NRPSs) and polyketide synthetases (PKSs) play a pivotal role in the production of bioactive natural products, such as antibiotics and cytotoxins. Despite biomedical and pharmaceutical importance, the molecular mechanisms and architectures of these multimodular enzyme complexes are not fully understood. Here, we report on an ABC transporter that forms a vital part of the nonribosomal peptide biosynthetic machinery. Emetic Bacillus cereus produces the highly potent, mitochondrial active nonribosomal depsipeptide cereulide, synthesized by the NRPS Ces. The ces gene locus includes, next to the structural cesAB genes, a putative ABC transporter, designated cesCD. Our study demonstrates that tethering of CesAB synthetase to the cell membrane by CesCD is critical for peptide assembly. In vivo studies revealed that CesAB colocalizes with CesCD on the cell membrane, suggesting direct involvement of this ABC transporter in the biosynthesis of a nonribosomal peptide. Mutation of cesCD, disrupting the assembly of the CesCD complex, resulted in decreased interaction with CesAB and, as a consequence, negatively affected cereulide biosynthesis. Specific domains within CesAB synthetase interacting with CesC were identified. Furthermore, we demonstrated that the structurally similar BerAB transporter from Bacillus thuringiensis complements CesCD function in cereulide biosynthesis, suggesting that the direct involvement of ABC transporter in secondary metabolite biosynthesis could be a widespread mechanism. In summary, our study revealed a novel, noncanonical function for ABC transporter, which is essential for megaenzyme functionality of NRPS. The new insights into natural product biosynthesis gained may facilitate the discovery of new metabolites with bioactive potential. IMPORTANCE This study revealed a novel, potentially conserved mechanism involved in the biosynthesis of microbial natural products, exemplified by the mitochondrial active depsipeptide cereulide. Similar to other bioactive substances, such as the last-resort antibiotics vancomycin and daptomycin, the antitumor drug cryptophycin or the cholesterol-lowering agent lovastatin, cereulide is synthesized nonribosomally by multienzyme machinery, requiring the concerted actions of multiple proteins to ensure correct product assembly. Given the importance of microbial secondary metabolites in human and veterinary medicine, it is critical to understand how these processes are orchestrated within the host cells. By revealing that tethering of a biosynthetic enzyme to the cell membrane by an ABC transporter is essential for nonribosomal peptide production, our study provides novel insights into synthesis of microbial secondary metabolites, which could contribute to isolation of novel compounds from cryptic secondary metabolite clusters or improve the yield of produced pharmaceuticals.

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